Heparin cofactor II (HCII) is a thrombin inhibitor found in relatively high concentration in plasma and in most tissues of the body. A member of the serpin superfamily, it is important to the prevention of thrombosis and arteriosclerosis. Heparin and dermatan sulfate accelerate its rate of inhibition 10[3] to 10[4]-fold through an allosteric mechanism that is only partly understood. This unique mechanism appears to involve binding of the N-terminus of HCII to exosite I of thrombin following the binding of glycosaminoglycans (GAG). Although some of the residues involved in this mechanism have been identified through site-directed mutagenesis, the structural changes that occur have not been identified. Another unusual feature of HCII is its active site sequence, which makes the serpin more sensitive to activation by particular GAG and therefore confers greater site specificity. This proposal seeks to use a combination of heteronuclear single quantum correlation (HSQC) NMR and fluorescence spectroscopy to elucidate in solution the sequence of events leading to HCII activation. A proposed model will be tested through the following specific aims 1) to determine the relative mobility of the N-terminus and its binding site(s), including the binding site of the hirudin-like acidic region, 2) to establish the activation mechanism by monitoring changes following the binding of heparin and dermatan sulfate, and 3) to determine whether the reactive center loop is partially inserted in beta-sheet A, and whether that insertion is affected by the binding of GAG.